PROJECT SUMMARY Translational control refers to the regulation of the protein synthesis machinery. Abnormality in translational control in humans has been implicated in many diseases, including cancer. Translational control in eukaryotes occurs predominantly during cap-dependent initiation, a multi-step process that is composed of: i) 43S ribosomal particle binding close to the 5' cap structure of a messenger RNA (mRNA); ii) 43S scanning toward the mRNA 3' end to locate the start of the coding region; and iii) the binding of the 60S large ribosomal subunit to 43S, which sets the stage for protein synthesis to begin. Kinetic characterization of cap-dependent initiation and its regulation during active translation and protein synthesis is limited due to the lack of appropriate kinetic approaches. We recently developed a single-molecule initiation assay that addressed this technical challenge. Our assay is based on cell extract with fluorescently labeled 43S and 60S ribosomal subunits. By measuring the real-time kinetics of 43S and 60S recruitment to single mRNA molecules, we can obtain important kinetic insights of the initiation process. The goal of the proposed research is to establish a quantitative understanding of cap-dependent initiation kinetics and to delineate the key molecular interactions and regulatory elements that control the initiation kinetics. In Aim 1, we will use our single-molecule observations to i) establish the kinetic scheme of 43S and 60S recruitment, ii) quantify the effect of translation-targeting chemicals, iii) interrogate the differential effect of m7G cap and polyA tail on stimulating 43S/60S binding, and iv) better define the roles of key initiation factors in ribosomal recruitment by depleting or mutating the factors in our extract. In Aim 2, we will measure 43S intrinsic scanning speed on structureless mRNA and the scanning slowing effect of mRNA structures. We will also seek a better understanding of the scanning mechanism by comparing the scanning kinetics of cap-dependent vs. m6A-mediated cap-independent initiation and by mutating eIF3. Lastly, we will study the function of helicases eIF4A and Ded1 in scanning by measuring the consequence of specific helicase depletion on the scanning kinetics for structured mRNAs. These two Aims will improve our understanding of the mechanism of cap-dependent initiation from the aspects of 43S/60S recruitment and scanning, and provide a kinetic perspective of initiation and its control, which is currently missing. The gained insights should facilitate a better understanding of translational control in a broad context.